Method for obtaining low ethanol-producing yeast strains, yeast strains obtained therefrom and their use

ABSTRACT

The present disclosure concerns a process for obtaining a variant yeast strain capable of producing less ethanol in an alcoholic fermentation process than its corresponding ancestral strain. The variant yeast strain is obtained by culturing the ancestral strain in the presence of increasing concentrations of a salt capable of causing an hyperosmotic stress to the ancestral yeast strain. The present disclosure also concerns variant yeast strain obtained from this process (for example the variant yeast strain deposited at Institut Pasteur, on Jan. 9, 2014, under accession number CNCM I-4832, the variant yeast strain deposited at Institut Pasteur, on Oct. 18, 2012 under accession number CNCM I-4684, the variant yeast strain deposited at Institut Pasteur, on Oct. 18, 2012 under accession number CNCM I-4685 and/or the variant yeast strain deposited at Institut Pasteur on Jan. 28, 2015 under accession number CNCM I-4952) as well as processes using the variant yeast strain (wine fermentation for example).

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

This application claims priority from EP patent application serialnumber 14290019.0 filed on Jan. 31, 2014. This application furtherincludes the following biological deposits (all made at InstitutPasteur) under accession number Collection Nationale des Cultures desMicroorganismes (CNCM) I-4832 (deposited on Jan. 9, 2014), CNCM I-4684(deposited on Oct. 18, 2012), CNCM I-4685 (deposited on Oct. 18, 2012)and CNCM I-4952 (deposited on Jan. 28, 2015). The content of thepriority application, the biological deposits and the sequence listingis herewith incorporated in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 240141_401C1_SEQUENCE_LISTING.txt. The text fileis 1.07 KB, was created on Sep. 29, 2020, and is being submittedelectronically via EFS-Web.

TECHNOLOGICAL FIELD

The present disclosure relates to a process for obtainingnon-genetically-modified variant yeast strains which have the ability ofproducing less ethanol than their corresponding ancestral/parentalstrains as well as variant yeast strains obtained or derived from thisprocess. The present disclosure also relates the use of such variantyeast strain during an alcoholic fermentation process, such as theproduction of wine.

BACKGROUND

Over the past twenty years, the alcohol content of wine has increasedconsiderably, by about 2% (v/v), as a result of the high sugar contentof the grapes currently used. This is mainly due to developments inwinemaking practices, with the harvest of very mature grapes beingfavored to adapt to consumer demand for rich and ripe fruit flavor inwine. This trend poses major problems for the wine industry. The marketis currently oriented towards beverages with moderate alcohol contents,in line with public prevention policies, consumer health issues andpreferences. In addition, as some countries impose taxes on the alcoholcontent, this trend raises economic issues. High levels of alcohol canalter the sensorial quality of wines, by increasing the perception ofhotness and, to a lesser extent, by decreasing the perception ofsweetness, acidity and aroma. Also, high ethanol levels generated duringfermentation may inhibit yeast activity and can lead to sluggish orstuck fermentations.

Consequently, reducing the ethanol content of wine has been a majorfocus of wine research, at various steps of the wine-making process.Several viticulture strategies are being developed to decrease sugaraccumulation on grapes. These approaches include the selection ofadequate grape varieties that accumulate less sugar and the modificationof culture techniques to reduce the berry sugar accumulation, such asirrigation, canopy management or limitation of photosynthesis. Physicaltechniques for de-alcoholisation, for example reverse osmosis,nano-filtration or distillation have also been developed and areavailable in the short-term. However, de-alcoholisation treatments areexpensive to implement, and may have detrimental effects on theorganoleptic quality of the wine.

An attractive and inexpensive option would be to use yeasts that produceless alcohol from the same amount of sugar. Indeed, there have been manyefforts to develop engineered wine yeast strains with reduced ethanolyield. One of the most efficient approaches was to divert metabolismtowards increased production of glycerol and thus away from ethanol. InSaccharomyces cerevisiae, glycerol plays major roles in redoxhomeostasis and in osmotic stress resistance: it is the main compatiblesolute in yeast. Glycerol is usually found in wines at concentrations inthe range 5 to 9 g/L and contributes positively to the quality of wineby providing body and sweetness. It may also confer viscosity at veryhigh concentrations (above 25 g/L), as in Botrytis wines. Reroutingcarbon towards glycerol led to a substantial decrease in ethanolproduction and accumulation of various compounds, including acetate andacetoin, both undesirable for wine sensorial quality. Rationalengineering of key reactions at the acetaldehyde branch point allowedthe accumulation of these undesirable compounds to be limited. Thisresulted in low alcohol strains being obtained in which the carbon fluxwas redirected towards glycerol and 2,3-butanediol, a polyol with nosensorial impact in wines. These engineered wine yeasts have thepotential to reduce the alcohol content of wine by 1 to 3% (v/v).However, the poor consumer acceptance of DNA recombinant technology infood is a major barrier to their commercialization. Consequently, thereis a great interest to use alternative, non-genetically modifiedorganism (GMO) approaches to improve the properties of wine yeaststrains.

It would be highly desirable to be provided with non-geneticallymodified variants yeast strains capable of producing less ethanol thantheir corresponding ancestral yeast strains. Preferably, the alcoholicfermentation of these variant yeast strain does not lead to theproduction of undesirable organoleptic properties in the fermentedproduct.

BRIEF SUMMARY

The present disclosure provides a process for obtaining a variant yeaststrain capable of producing, when compared to an ancestral yeast strain,more glycerol and less ethanol during an alcoholic fermentation process.The process relies on the use of a salt capable of causing anhyperosmotic stress to the ancestral yeast strain as well as culturingthe yeast strain in a high concentration of a carbon source untilexhaustion of the carbon source. Surprisingly, the variant yeast strainobtained produce more glycerol and less ethanol than the ancestral yeaststrain from which they are derived, even in the absence of the salt. Inan embodiment, the variant yeast strain is not more resistant to thehyperosmotic stress than its corresponding ancestral yeast strain, butdisplays increased viability and a gain of fitness in carbon starvationconditions when compared to its ancestral yeast strain. In someembodiments, the variant yeast strain (when compared to the ancestralyeast strain) produces the same amount or less of acetate and/or acetoinduring an alcoholic fermentation.

According to a first aspect, the present disclosure provides a processfor obtaining a variant yeast strain capable of producing, when comparedto an ancestral yeast strain, more glycerol and less ethanol in analcoholic fermentation process. Broadly, the process comprises a)culturing the ancestral yeast strain in a first culture mediumcomprising a salt capable of causing an hyperosmotic stress to theancestral yeast strain, wherein the ancestral yeast strain is culturedin increasing salt concentrations and under conditions to achieveglucose depletion in the first culture medium so as to obtain a firstcultured yeast strain; and b) culturing the first cultured yeast strainin a second culture medium comprising the salt, wherein the firstcultured yeast strain is cultured at a fixed salt concentration andunder conditions to achieve glucose depletion in the second culturemedium so as to obtain the variant yeast strain. The salt used in theprocess has a countercation which is different than a sodium cation. Inthe process, the concentration of the salt in the second culture mediumis higher than the concentration of the salt in the first culturemedium. In an embodiment, the concentration of the salt in the firstculture medium is between about 1.25 M and less than about 1.9 M orabout 2.4 M. In another embodiment, the concentration of the salt in thesecond culture medium is at least about 2.4 M. In an embodiment, theprocess further comprises, at step a), increasing the salt concentrationweekly or monthly. In still another embodiment, the first culture mediumcomprises glucose and the process further comprises, at step a),culturing the ancestral yeast strain in the first culture medium whiledecreasing glucose concentrations. In such embodiment, the concentrationof glucose can be decreased weekly or monthly. Further, still in suchembodiment, the concentration of glucose in the first culture medium canbe between about 14.0% and about 8.0% (w/v) or between about 14.0% andabout 9.6% (w/v) with respect to the total volume of the first culturemedium. In another embodiment, the second culture medium comprisesglucose and the process further comprises, at step b), culturing thefirst cultured yeast at a fixed glucose concentration. In suchembodiment, the fixed glucose concentration of the second culture mediumis preferably 8.0% (w/v) with respect to the total volume of the secondculture medium. In an embodiment, the process can further comprisemating haploid spores of the variant yeast strain to obtain a varianthybrid strain. In still another embodiment, the salt has a potassiumcation, such as, for example, KCl. In yet another embodiment, theancestral and/or variant yeast strain is from a Saccharomyces speciesand preferably from a genus selected from the group consisting ofSaccharomyces arboricolus, Saccharomyces eubayanus, Saccharomycesbayanus, Saccharomyces cerevisiae, Saccharomyces kudriadzevii,Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomycespastorianus, Saccharomyces carsbergensis, Saccharomyces uvarum andinter-species hybrids.

According to a second aspect, the present disclosure provides a variantyeast strain capable of producing, when compared to an ancestral yeaststrain, more glycerol and less ethanol in an alcoholic fermentationprocess. In an embodiment, the variant yeast is obtained by the processdescribed herein. In an embodiment, the variant yeast strain obtainedcan be used for making a fermented product, such as wine (e.g., redwine) or beer. In an embodiment, the variant yeast strain is at leastone of the one deposited at Institut Pasteur, on Jan. 9, 2014, underaccession number Collection Nationale des Cultures des Microorganismes(CNCM) I-4832, the one deposited at Institut Pasteur, on Oct. 18, 2012under accession number Collection Nationale des Cultures desMicroorganismes (CNCM) I-4684 and the one deposited at Institut Pasteur,on Oct. 18, 2012 under accession number Collection Nationale desCultures des Microorganismes (CNCM) I-4685, on Jan. 28, 2015 underaccession number Collection Nationale des Cultures des Microorganismes(CNCM) I-4952 as well as any combination thereof.

According to a third aspect, the present disclosure provides a processfor making a fermented product. Broadly, the process comprisescontacting the variant yeast strain described herein with a fermentablesource of nutrients. In an embodiment, the fermented product is wine(e.g., red wine) and the fermentable source of nutrients is a grapemust. In another embodiment, the fermented product is beer and thefermentable source of nutrient is starch (e.g., derived from cerealssuch as barley for example).

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings, showing by way ofillustration, a preferred embodiment thereof, and in which:

FIGS. 1A-1D shows glycerol concentration (bars providing the minimal andmaximal concentration) and ethanol yield (black lozenges with the errorbars providing minimal and maximal yields) (A, B), and glycerolconcentration (bars) and residual glucose after 15 (white triangles withthe error bars providing maximal and minimal concentrations) and 30(black triangles with the error bars providing maximal and minimalconcentrations) days of fermentation (C, D) for the ancestral strain(EC1118), evolved populations (dark grey, label starting with “PK”) andisolates (evolved strains) (light grey, label starting with “K”) fromthe independent lineages a (A, C) and b (B, D). Fermentations werecarried out in 300 mL MS210, 260 g/L glucose, at 28° C. in triplicate.The number in the different labels refers to the number of generationsthat the populations and strains were submitted to adaptive evolution.

FIG. 2 shows the selective advantage of the evolved strains. Viabilityof EC1118 (●, black line), K300.2(a) (▴, dark grey line) and K300.1(b)(∘, light gray line) during culture in YPD+8% glucose and 2.4 M KCl at28° C. Results are shown as the number (×10⁷) of living cells/mL infunction of time (hours). Sugar exhaustion is observed after 100 hours.Each point includes the measured value as well as the standarddeviation.

FIGS. 3A-3B illustrates the fermentation performances (A) and cellpopulation (B) of the ancestral strain EC1118 (●, black line) and theevolved strains K300.2(a) (▴, dark grey line) and K300.1(b) (∘, lightgrey line) on MS210 medium, 260 g/L glucose, at 28° C. Results in panelA are provided as dCO₂/dt (g/l/H) in function of time (hours). Resultsin panel B are provided as the number of cells (×10⁷)/mL in function oftime (hours) and include the standard deviation.

FIGS. 4A-4C shows by-product yields for strains EC1118 and K300.1(b).Metabolites were measured after 30 days of fermentation in 300 mL ofMS210, 260 g/L glucose at 16° C., 20° C., 24° C., 28° C., 32° C. and 34°C. Results are shown for ethanol (A, provided as g/g of consumed glucosein function of time and of strain used), glycerol (B, provided as g/g ofconsumed glucose in function of time and strain used) and succinate (C,provided as g/g of consumed glucose in function of time and strainused). Each points includes the measured valued as well as the standarddeviation.

FIG. 5 illustrates the kinetics of wine fermentation on Grenache forEC1118 (black line) and K300.1(b) (dark grey line). 72 mg/L nitrogen (15g/hL of DAP and 30 g/hL of Fermaid®E) were added at the time pointindicated by an arrow. Results are shown as dCO₂/dt(g/L/H) in functionof time (hours). Each points includes the measured valued as well as thestandard deviation.

FIG. 6 illustrates the kinetics of wine fermentation trial N2 onsynthetic must containing 235 g/L sugars for the ancestral strain(EC1118) and an H2 generation hybrid. Results are shown asdCO₂/dt(g/L/H) in function of time (hours).

FIG. 7 illustrates the kinetics of wine fermentation trial N3 onsynthetic must containing 260 g/L sugars for the ancestral strain(EC1118) and an H2 generation hybrid. Results are shown asdCO₂/dt(g/L/H) in function of time (hours).

FIG. 8 illustrates the kinetics of wine fermentation trial N4 on a Syrahvariety grape must for the ancestral strain (EC1118) and an H2generation hybrid (120-A5). Results are shown as dCO₂/dt(g/L/H) infunction of time (hours). Arrows indicated when oxygen was added to thefermentation.

DETAILED DESCRIPTION

An attractive and inexpensive option to obtain wine having a loweralcohol content would be to use yeasts that produce less alcohol fromthe same amount of sugar. Indeed, there have been many efforts todevelop engineered wine yeast strains with reduced ethanol yield. One ofthe most efficient approaches was to divert metabolism towards increasedproduction of glycerol and thus away from ethanol. In Saccharomycescerevisiae, glycerol plays major roles in redox homeostasis and inosmotic stress resistance: it is the main compatible solute in yeast.Glycerol is usually found in wines at concentrations in the range 5 to 9g/L and contributes positively to the quality of wine by providing bodyand sweetness. It may also confer viscosity at very high concentrations(above 25 g/L), as in Botrytis wines. Usually, rerouting carbon towardsglycerol led to a substantial decrease in ethanol production andaccumulation of various compounds, including acetate and acetoin, bothundesirable for wine sensorial quality. Rational genetic engineering ofkey reactions at the acetaldehyde branch point allowed the accumulationof these undesirable compounds to be limited. This resulted in lowalcohol strains being obtained in which the carbon flux was redirectedtowards glycerol and 2,3-butanediol, a polyol with no sensorial impactin wines. These engineered wine yeasts have the potential to reduce thealcohol content of wine by 1 to 3% (v/v). However, the poor consumeracceptance of DNA recombinant technology in food is a major barrier totheir commercialization. Consequently, there is a great interest to usealternative, non-GMO approaches to improve the properties of wine yeaststrains.

Process for Obtaining Low Ethanol-Producing Variant Yeast Strains

Adaptive laboratory evolution (ALE) experiments, based on long termadaptation of yeast under environmental or metabolic constraints, hasbeen used to improve yeast strains for biotechnological applications,including wine-making. Experimental evolutions using sodium chloride togenerate osmotic stress have been used to study evolutionary processes,and in more applied work, to increase the tolerance of baking strains tofreezing. NaCl-resistant evolved industrial strains were obtained, butthe production of glycerol and ethanol by the evolved strains was notaffected.

The present disclosure provides a process for obtaining a variant yeaststrain. In the context of the present disclosure, a “variant yeaststrain” is a natural (e.g., not genetically modified using recombinantDNA/RNA technology) yeast strain mutant which has been selected from an“ancestral” yeast strain using ALE (based on the salt described herein)to redirect carbon flux towards glycerol and, ultimately, reduce theproduction of ethanol during alcoholic fermentation. The ancestral yeaststrain and the variant yeast strain are non-genetically modifiedorganisms, e.g., their genomic content has not been altered by theintroduction of exogenous nucleic acid molecules or the removal ofendogenous nucleic acid molecules using genetic engineering techniques.In some embodiments, the alcoholic strength by volume (% v/v) of afermented product (e.g., wine) obtained with the variant yeast strain isreduced when compared to the alcoholic strength by volume of a fermentedproduct (e.g., wine) obtained with the ancestral yeast strain, bybetween about 0.40% and 2.00% or by at least 0.40%, 0.45%, 0.50%, 0.60%,0.70%, 0.80%, 0.90%, 1.00%, 1.10%, 1.20%, 1.30%, 1.40%, 1.50%, 1.60%,1.70%, 1.80%, 1.90% or 2.00%. In alternative or complimentaryembodiments, the ratio of the glycerol content of a fermented product(e.g., wine) obtained with the variant yeast strain to the glycerolcontent of a fermented product (e.g., wine) obtained with the ancestralyeast strain, is between 1.25 and 2.40 or at least 1.25 1.30, 1.35,1.40, 1.45, 1.50, 1.55, 1.60, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00,2.10, 2.15, 2.20, 2.25, 2.30, 2.35 or 2.40.

In the context of the present disclosure, during an alcoholicfermentation process, the “variant” yeast strain does not produce anamount of acetate, acetaldehyde and acetoin which can alter theorganoleptic properties of the fermented product. In an embodiment, thecontent of acetate, acetaldehyde or acetoin in the fermented productobtained by using the variant yeast strain is either equal to or lessthan the corresponding content of acetate, acetaldehyde or acetoin inthe fermented product obtained by using the ancestral yeast strain. Inembodiments, the content of acetate, acetaldehyde or acetoin in thefermented product obtained by using the variant yeast strain isaugmented when compared to the corresponding fermented product obtainedusing the ancestral yeast strain, this increase is equal to or less than70%. In still some embodiments, the variant yeast strain can produce agreater amount of one or more compounds (such as 2,3-butanediol), whencompared to the ancestral yeast strain, which does not impact theorganoleptic properties of the fermented product. In some embodiments,the variant yeast strains are not more resistant to an hypersomoticshock caused by the salt than the ancestral yeast strain, but thevariant yeast strains display better viability and a gain of fitness(when compared to the ancestral yeast strain) under conditions ofhyperosmotic stress and carbon starvation.

In order to obtain the variant yeast strain, an ancestral yeast strainis submitted to ALE and is cultured in increasing salt concentrations.The salt used during ALE is capable of causing an hyperosmotic stress tothe ancestral yeast strain. In the context of the present disclosure,the term hyperosmotic stress (also referred to as an hyperosmotic shock)is an increase in the solute (e.g., ionic) concentration around a yeastcell causing a rapid change in the movement of water across its cellmembrane. In such conditions, an inhibition of the transport ofsubstrates and cofactors into the cell can occur thus causing a shock.The salt in ALE can either be a single type of salt or a combination ofsalts capable of causing an hyperosmotic shock. The salt or thecombination of salts used in the ALE described herein must be capable ofproviding a specific osmolality to the culture medium without inducingtoxicity towards the parental strain. For example, in the context of thepresent disclosure, the cation of the salt (or combination of salts)used in ALE lacks toxicity with respect to the ancestral yeast strainwhen used at a concentration for providing an initial osmolality of atleast 1 500 mmol/kg, at least 1 600 mmol/kg, at least 1 700 mmol/kg, atleast 1 800 mmol/kg, at least 1 900 mmol/kg, at least 2 000 mmol/kg, atleast 2 100 mmol/kg or at least 2 200 mmol/kg. The salt used in theprocesses described herein has a countercation which is different thansodium. For example, the salt can have a potassium countercation. Suchsalts include, but are not limited to KCl. Such salts exclude NaCl whosecation has been shown to cause toxicity to the ancestral yeast strain.Such salts also exclude sulfites, such as sodium sulfite (Na₂SO₃), whichgenerate sodium cations and provide yeasts strains only modestly capableof decreasing the alcohol by volume content of fermented products.

In a first step, the process for obtaining the variant yeast strainincludes culturing yeast strains in increasing salt concentrations. Theancestral yeast strain is used to inoculate (at a predetermined amount)a culture medium containing the salt at a specific concentration. Theancestral yeast strain is then cultured under conditions so as toachieve carbon or glucose depletion (e.g. also referred to as carbonstarvation). Then, a pre-determined amount of the cultured yeasts isused to inoculate a fresh medium containing either the same saltconcentration or a higher salt concentration. This cycle is repeateduntil the cultured yeasts reach a relatively stable phenotype withrespect to glycerol and ethanol production in alcoholic fermentation. Insome embodiments, this cycle is repeated for at least (about) 200, 250,300, 250, 400, 450, 500, 550, 600, 650, 700 or 750 yeast generations.During the process, the osmolality of the medium used to culture theyeasts is progressively increased from about 1 5000 to about 5 000mmol/kg. For example, in an embodiment, the osmolality during theinitial phase of the process can be (about) at least 1 500 mmol/kg, atleast 1 600 mmol/kg, at least 1 700 mmol/kg, at least 1 800 mmol/kg, atleast 1 840 mmol/kg, at least 1 900 mmol/kg, at least 2 000 mmol/kg, atleast 2 100 mmol/kg, at least 2 105 mmol/kg or at least 2 200 mmol/kg.Alternatively or in combination, the osmolality during the final phaseof the first step of the process can be (about) at most 4 800 mmol/kg,at most 4 740 mmol/kg, at most 4 700 mmol/kg, at most 4 600 mmol/kg, atmost 4 500 mmol/kg, at most 4 400 mmol/kg, at most 4 300 mmol/kg, atmost 4 200 mmol/kg, at most 4 100 mmol/kg, at most 4 000 mmol/kg, atmost 3 900 mmol/kg, at most 3 800 mmol/kg, at most 3 730 mmol/kg, atmost 3 700 mmol/kg, at most 3 600 mmol/kg or at most 3 500 mmol/kg. Inan embodiment, the osmolality during the first step of the process isincreased from (about) 1 500 mmol/kg to (about) 4 800 mmol/kg, 4 740mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg,3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600 mmol/kg or 3 500mmol/kg. In another embodiment, the osmolality during the process isincreased from (about) 1 600 mmol/kg to (about) 4 800 mmol/kg, 4 740mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg,3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600 mmol/kg or 3 500mmol/kg. In yet another embodiment, the osmolality during the first stepof the process is increased from (about) 1 700 mmol/kg to (about) 4 800mmol/kg, 4 740 mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4400 mmol/kg, 4 300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg,3 900 mmol/kg, 3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600mmol/kg or 3 500 mmol/kg. In still a further embodiment, the osmolalityduring the first step of the process is increased from (about) 1 800mmol/kg to (about) 4 800 mmol/kg, 4 740 mmol/kg, 4 700 mmol/kg, 4 600mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4 300 mmol/kg, 4 200 mmol/kg, 4100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg, 3 800 mmol/kg, 3 730 mmol/kg,3 700 mmol/kg, 3 600 mmol/kg or 3 500 mmol/kg. In another embodiment,the osmolality during the first step of the process is increased from(about) 1 840 mmol/kg to (about) 4 800 mmol/kg, 4 740 mmol/kg, 4 700mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4 300 mmol/kg, 4200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg, 3 800 mmol/kg,3 730 mmol/kg, 3 700 mmol/kg, 3 600 mmol/kg or 3 500 mmol/kg. In still afurther embodiment, the osmolality during the first step of the processis increased from (about) 1 900 mmol/kg to (about) 4 800 mmol/kg, 4 740mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg,3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600 mmol/kg or 3 500mmol/kg. In another embodiment, the osmolality during the first step ofthe process is increased from (about) 2 000 mmol/kg to (about) 4 800mmol/kg, 4 740 mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4400 mmol/kg, 4 300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg,3 900 mmol/kg, 3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600mmol/kg or 3 500 mmol/kg. In yet another embodiment, the osmolalityduring the first step of the process is increased from (about) 2 100mmol/kg to (about) 4 800 mmol/kg, 4 740 mmol/kg, 4 700 mmol/kg, 4 600mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4 300 mmol/kg, 4 200 mmol/kg, 4100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg, 3 800 mmol/kg, 3 730 mmol/kg,3 700 mmol/kg, 3 600 mmol/kg or 3 500 mmol/kg. In still a furtherembodiment, the osmolality during the first step of the process isincreased from (about) 2 105 mmol/kg to (about) 4 800 mmol/kg, 4 740mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4 400 mmol/kg, 4300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg, 3 900 mmol/kg,3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600 mmol/kg or 3 500mmol/kg. In another embodiment, the osmolality during the first step ofthe process is increased from (about) 2 200 mmol/kg to (about) 4 800mmol/kg, 4 740 mmol/kg, 4 700 mmol/kg, 4 600 mmol/kg, 4 500 mmol/kg, 4400 mmol/kg, 4 300 mmol/kg, 4 200 mmol/kg, 4 100 mmol/kg, 4 000 mmol/kg,3 900 mmol/kg, 3 800 mmol/kg, 3 730 mmol/kg, 3 700 mmol/kg, 3 600mmol/kg or 3 500 mmol/kg. In still another embodiment, the osmolalityduring the first step of the process is increased from (about) 1 500mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1 840 mmol/kg, 1900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kg or 2 200mmol/kg to (about) 4 800 mmol/kg. In still another embodiment, theosmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 740 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 700 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 600 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 500 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 400 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 300 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 200 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 100 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 4 000 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 3 900 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 3 800 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 3 730 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 3 700 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 3 600 mmol/kg. In still another embodiment,the osmolality during the first step of the process is increased from(about) 1 500 mmol/kg, 1 600 mmol/kg, 1 700 mmol/kg, 1 800 mmol/kg, 1840 mmol/kg, 1 900 mmol/kg, 2 000 mmol/kg, 2 100 mmol/kg, 2 105 mmol/kgor 2 200 mmol/kg to (about) 3 500 mmol/kg.

Broadly, the process comprises at least two phases. In a first phase,the yeasts are cultured at a relatively low salt concentration which isincreased during culture. In a second phase, the yeasts are cultured ata higher and fixed salt concentration. During the process, the initialcarbon source concentration in the culture medium (e.g., prior toculture) is high (at least 8% (w/v) with respect to total volume of theculture medium) in the culture medium so as to maintain the fermentativeperformances of the cultured yeast in the presence of, initially, arelatively high concentration of carbon. In the context of the presentdisclosure, in both phases, the yeasts are cultured until the carbonsource is depleted (e.g., until a state of carbon starvation is reached)from the culture medium prior to proceeding to a further inoculationinto a fresh medium.

During the first phase of the process, the yeasts are cultured in afirst culture medium containing the salt and the carbon source. In thecontext of the present disclosure, the “first culture medium” refers toa culture medium that is used during the first phase of the process. Thefirst culture medium can be any type of medium suitable for the growthof yeasts. Even though the first culture medium can be a solid medium,the first culture medium is preferably a liquid medium. Yeast-adaptedmedium include, but are not limited to, the Yeast Peptone Dextrose (YPD)medium or a defined/synthetic SD medium based a yeast nitrogen basemedium. Optionally, the first culture medium can be supplemented withbacto-yeast extract and bactopeptone.

Initially, the concentration of the salt in the first culture medium isselected to increase the osmolality of the first culture medium (e.g.,to reach at least at least 1 500 mmol/kg, at least 1 600 mmol/kg, atleast 1 700 mmol/kg, at least 1 800 mmol/kg, at least 1 840 mmol/kg, atleast 1 900 mmol/kg, at least 2 000 mmol/kg, at least 2 100 mmol/kg, atleast 2 105 mmol/kg or at least 2 200 mmol/kg) and cause a reduction ingrowth (when compared to a yeast in the same medium without the salt) ofthe ancestral strain (which has not previously been cultured in thepresence of such high salt concentration). This reduction in growth canbe, for example, at least about 1.5, 2.0, 3.0, 4.0 fold or even more,when compared to the ancestral yeast strain cultured in similarconditions but in the absence of the salt. This salt concentration cancorrespond to 1.25 M when KCl is used as the salt to supplement a YPDmedium and it provides an osmolality of about 2 105 mmol/kg. Thereafter,the yeasts are cultured in increasing salt concentrations. During thefirst phase, the salt concentration can be between about 1.25 M to lessthan about 2.4 M or at least about 1.25 M, 1.30 M, 1.40 M, 1.50 M, 1.60M, 1.70 M, 1.80 M, 1.90 M, 2.0 M, 2.1 M, 2.2 M or 2.3 M. In anembodiment, the salt concentration in the first culture medium canserially be increased from about 1.25 M to about 1.30 M, from about 1.30M to about 1.40 M, from about 1.40 M to about 1.50 M, from about 1.50 Mto about 1.60 M, from about 1.60 M to about 1.70 M, from about 1.70 M toabout 1.80 M, from about 1.80 M to about 1.90 M, from about 1.9 M toabout 2.0 M, from about 2.0 M to about 2.1 M, from about 2.1 M to about2.2 M, from about 2.2 M to about 2.3 M and from about 2.3 M to about 2.4M. This serial increase can be made at pre-determined intervals, forexample at weekly intervals or at monthly intervals. In someembodiments, the first phase can comprise two sub-phases: a firstsub-phase in which the salt concentration is increased weekly (forexample by increasing the salt concentration from about 1.25 M to about1.9 M) and a second sub-phase in which the salt concentration isincreased monthly (for example by increasing the salt concentration fromabout 1.9 M to about 2.4 M).

The first culture medium also comprises an available carbon source, suchas glucose. The initial concentration of the carbon source in the firstculture medium is selected to allow the maintenance of good fermentativeperformances of the yeasts. For example, prior to the culture with theyeasts, the concentration of the carbon source in the first culturemedium is at least 8% (w/v), between about 8% and about 14% (w/v) orbetween about 9.6% (w/v) and about 14% (w/v) with respect to the totalvolume of the culture medium. During the first phase, the initial carbonsource concentration can be at least about 8.0%, 8.4%, 8.8%, 9.2%, 9.6%,10.0%, 10.4%, 10.8%, 11.2%, 11.6%, 12.0%, 12.4%, 12.8%, 13.2%, 13.6% or14%, preferably at least about 9.6%, 10.0%, 10.4%, 10.8%, 11.2%, 11.6%,12.0%, 12.4%, 12.8%, 13.2%, 13.6% or 14.0% (w/v with respect to thetotal volume of the culture medium). In an embodiment, the carbon sourceconcentration in the first culture medium can serially be decreased fromabout 14.0% to about 13.6%, from about 13.6% to about 13.2%, from about13.2% to about 12.8%, from about 12.8% to about 12.4%, from about 12.4%to about 12.0%, from about 12.0% to about 11.6%, from about 11.6% toabout 11.2%, from about 11.2% to about 10.8%, from about 10.8% to about10.4%, from about 10.4% to about 10.0%, from about 10.0% to about 9.6%,from about 9.6% to about 9.2%, from about 9.2% to about 8.8%, from about8.8% to about 8.4% and from about 8.4% to about 8.0%. This serialdecrease can be made at pre-determined intervals, for example at weeklyintervals or at monthly intervals.

In some embodiments, the first phase can comprise two sub-phases: afirst sub-phase in which the carbon source concentration is decreasedweekly (for example by decreasing the carbon concentration from about14% to about 9.6%) and a second sub-phase in which the carbon sourceconcentration is increased monthly (for example by decreasing the carbonsource concentration from about 9.6% to about 8.0%).

At the initial step of the first phase, the ancestral yeast strain canbe first inoculated at a pre-determined concentration (e.g., an OD₆₀₀ of1.0 for example) in the first culture medium. The ancestral strain iscultured under conditions so as to allow yeast growth (e.g., 28° C.under agitation). The yeasts are cultured in the first culture mediumuntil the carbon source (usually glucose) has been metabolized (e.g.,depleted). The time to reach carbon depletion will depend on the type ofculture medium used, the amount of yeasts used to inoculate the culturemedium, the incubation conditions as well as the initial amount of thecarbon source. However, after about 4 to 7 generations (e.g., about aweek), the carbon source in a YPD medium supplemented with 8% (w/w)glucose and inoculated at an OD₆₀₀ of 1.0 with cultured yeasts isconsidered depleted. In another example, after about 8 to 14 generations(e.g., about two weeks), the carbon source in a YPD medium supplementedwith 14% (w/w) glucose and inoculated at an OD₆₀₀ of 1.0 with culturedyeasts is considered depleted.

During the first phase, once the carbon source has been depleted fromthe culture medium, the cultured yeasts can be maintained in a carbonstarvation phase or can be inoculated into a fresh medium containing ahigher salt concentration and a further source of available carbon.During the first phase, the increase in salt concentration between twoculture media can be, for example, 0.05 M (initially) and 0.1 M(afterwards). In some embodiments, a more or less important increase insalt concentration can be made to achieve similar results. The firstphase is maintained for at least about 175 days, at least about 25 weeksor at least about 100 generations. In an embodiment, the first phase ismaintained until the growth rate of the cultured yeasts increases by atleast about 5%, 6%, 7%, 8%, 9% or 10% when compared to the growth rateof the cultured yeasts at the initiation of the first phase (when areduction in growth rate is observed because of the presence of thesalt).

When the salt concentration is increased in the first culture medium, acorresponding glucose concentration can be decreased in the firstculture medium. For example, in an embodiment, when the saltconcentration is increased by 0.1 M in the first culture medium, theglucose concentration can be decreased (prior to the culture) by 0.4%(w/v) with respect to the total volume of the first culture medium. Instill another embodiment, when the salt concentration is increased byabout 0.05 M in the first culture medium, the glucose concentration(prior to the culture) is decreased by about 0.2% (w/v) with respect tothe total volume of the first culture medium.

Once the first phase of the process has been completed, in a secondphase, the yeasts are cultured in a second culture medium containing thesalt and the carbon source. In the context of the present disclosure,the “second culture medium” refers to a culture medium that is usedduring the second phase of the process. The second culture medium can beany type of medium suitable for the growth of yeasts. Even though thesecond culture medium can be a solid medium, the second culture mediumis preferably a liquid medium. Yeast-adapted medium include, but are notlimited to, the Yeast Peptone Dextrose (YPD) medium or adefined/synthetic SD medium based a yeast nitrogen base medium.Optionally, the second culture medium can be supplemented withbacto-yeast extract and bactopeptone.

During the second phase, the second culture medium has a saltconcentration that is the same or higher than the first culture mediumat the end of the first phase. However, during the second phase, thesalt concentration remains the same and does not increase. In anembodiment, the salt concentration of the second culture medium can beabout 2.4 M. In some embodiment, the second culture medium has anosmolality of at most about 4 800 mmol/kg, at most about 4 740 mmol/kg,at most about 4 700 mmol/kg, at most about 4 600 mmol/kg, at most about4 500 mmol/kg, at most about 4 400 mmol/kg, at most about 4 300 mmol/kg,at most about 4 200 mmol/kg, at most about 4 100 mmol/kg, at most about4 000 mmol/kg, at most about 3 900 mmol/kg, at most about 3 800 mmol/kg,at most about 3 730 mmol/kg, at most about 3 700 mmol/kg, at most about3 600 mmol/kg or at most about 3 500 mmol/kg. mmol/kg.

As indicated above, the second culture medium also comprises a carbonsource, such as glucose. The initial concentration of the carbon sourcein the second culture medium is selected to allow the maintenance ofgood fermentative performances of the yeasts. In an embodiment, theinitial concentration of the carbon source in the second culture mediumis equal to or lower than the initial concentration of the carbon sourcein the first culture medium at the end of the first phase of theprocess. Further, the initial glucose concentration (prior to culture)in the second culture medium remains the same during the second phase.In an embodiment, prior to the culture with the yeasts, theconcentration of glucose in the second culture medium is between about8% and about 14% (w/v), preferably between about 8% and about 10% (w/v)and even more preferably about 8% (w/v with respect to the total volumeof the second culture medium). In an embodiment, the salt concentrationof the second culture medium is about 2.4 M. When KCl is used at suchconcentration to supplement a YPD medium, this corresponds to anosmolality of about 3 730 mmol/kg.

During the second phase, the first cultured yeast strain (e.g., a yeaststrain that has been submitted and completed the first phase of theprocess) is first inoculated at a pre-determined concentration (e.g. anOD₆₀₀ of 1.0) in the second culture medium containing the salt as wellas the carbon source. The yeast strain is cultured under conditions soas to allow yeast growth (e.g., 28° C. under agitation). The yeasts arecultured in the second culture medium until the carbon source (usuallyglucose) has been metabolized (e.g., depleted). The time to reach carbondepletion will depend on the type of culture medium used, the amount ofyeasts used to inoculate the culture medium, the incubation conditionsas well as the initial amount of the carbon source. However, after about4 to 7 generations (e.g., about a week), the carbon source in a YPDmedium supplemented with about 8% (w/w) glucose and inoculated at anOD₆₀₀ of 1.0 with cultured yeasts is considered depleted.

During the second phase, once the carbon source has been depleted fromthe culture medium, the cultured yeasts are either maintained in aglucose starvation state or inoculated into a fresh medium containingthe same salt concentration and the same carbon source concentrationthan the previous medium. During the second phase, the saltconcentration can be about 2.4 M and the glucose concentration can beabout 8.0% (w/w). In embodiments, the second phase is maintained for atleast about 553 days, at least about 79 weeks and/or at least about 200generations. In an embodiment, the second phase lasts until the culturedyeast strain exhibit a stable phenotype with respect to glycerol andethanol production in the absence of the salt stress.

The first and second culture medium can have the same base medium anddiffer only with respect to the salt, the salt concentration, the carbonsource and/or the carbon source concentration. Alternatively, the firstand second can have different base medium.

At the end of the second phase of the process, it is expected that thecultured yeast strains (now referred to as variant yeasts strains) havethe ability of producing more glycerol during an alcoholic fermentationthan the ancestral yeast strain. For example, in some embodiments, theratio of the glycerol content of a fermented product (e.g., wine)obtained with variant yeast strains to the glycerol content of afermented product (e.g., wine) obtained with the ancestral yeast strain,is between 1.25 and 2.40 or at least about 1.25, 1.30, 1.35, 1.40, 1.45,1.50, 1.55, 1.60, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.10, 2.15,2.20, 2.25, 2.30, 2.35 or 2.40. It is also expected that the variantyeast strains have the ability of producing less ethanol during analcoholic fermentation than the ancestral yeast strain. For example, insome embodiments, the alcoholic strength by volume (% v/v) of afermented product (e.g., wine) obtained with the variant yeast strain isreduced, when compared to the alcoholic strength by volume of afermented product (e.g., wine) obtained with the ancestral yeast strain,by at least 0.40% or between about 0.40% and 2.00% or by at least about0.40%, 0.45%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90%, 1.00%, 1.10%, 1.20%,1.30%, 1.40%, 1.50%, 1.60%, 1.70%, 1.80%, 1.90% or 2.00%. In someembodiments, the variant yeast strain can produce a greater amount ofone or more compounds (such as 2,3-butanediol), when compared to theancestral yeast strain, which does not impact the organolepticproperties of the fermented product.

The variant yeasts strains can optionally be further submitted toconventional breeding (which excludes genetic engineering manipulations)to further increase their ability to produce glycerol, decrease theirability to produce ethanol during an alcoholic fermentation and/orproduce inter-species hybrid having a similar phenotype. Conventionalbreeding conducted with yeasts of the same species (e.g., intra-speciesbreeding) or with yeasts of different species (e.g., inter-speciesbreeding). Such breeding techniques are known to those skilled in theart and usually include (i) the production of haploid yeast spores froma selected variant yeast strain, (ii) the selection of haploid strainshaving the desired phenotype (e.g., an increased capacity in producingglycerol and/or a decreased capacity of producing ethanol during analcoholic fermentation for example) and (iii) the mating of the selectedhaploid strains to obtain stable hybrid (e.g., diploid) strain and theselection of a hybrid strain having the desired phenotype (e.g., anincreased capacity in producing glycerol, a decreased capacity ofproducing ethanol during an alcoholic fermentation and/or aninter-species hybrid having the desired (stable) phenotype). Thisoptional breeding step can be used to obtain 1^(st) generation hybrids(e.g., usually referred to as H1), 2^(nd) generation hybrids (e.g.,usually referred to as H2) and even 3^(rd) generation hybrids (e.g.,usually referred to as H3). As indicated above, the breeding step caninclude the generation of intra-species and inter-species hybrids).

Prior to or after the breeding, the variant yeast strain can optionallybe submitted to a further step for determining their ability to conductcellular respiration. Variant yeast strains capable of cellularrespiration are considered to be useful for wine-making applications.

The process described herein can be apply to yeasts and is especiallyuseful for the generation of variant yeast strains destined to be usedin alcoholic fermentations. Exemplary yeasts includes, but are notlimited to Saccharomyces sp. (for example, from the genus Saccharomycesarboricolus, Saccharomyces eubayanus, Saccharomyces bayanus,Saccharomyces cerevisiae, Saccharomyces kudriadzevii, Saccharomycesmikatae, Saccharomyces paradoxus, Saccharomyces pastorianus,Saccharomyces carsbergensis and Saccharomyces uvarum.), Brettanomycessp. (Teleomorph Dekkera sp.), Candida (Teleomorphs for different speciesfrom several genera including Pichia sp., Metschnikowia sp.,Issatchenkia sp., Torulaspora sp. and Kluyveromyces sp.), Kloeckera sp.(Teleomorph Hanseniaspora sp.), Saccharomycodes sp., Schizosaccharomycessp. and/or Zygosaccharomyces sp as well as inter-species hydrids derivedfrom any one of these yeast species.

Variant Yeast Strains and their Use in Alcoholic Fermentation

The present disclosure also concerns the variant yeast strain obtainedby the process described herein. As described herein, the “variant yeaststrain”, during an alcoholic fermentation, produces more glycerol andless ethanol than its corresponding ancestral yeast strain and isobtained by the process described herein. As such, the fermentedproducts obtained using the variant yeast strain has less ethanol thanthe fermented products obtained using the ancestral yeast strain. Forexample, the alcoholic strength by volume (% v/v) of a fermented product(e.g., wine) obtained with the variant yeast strain can be reduced, whencompared to the alcoholic strength by volume of a fermented product(e.g., wine) obtained with the ancestral yeast strain, by between about0.40% and about 2.00% or by at least about 0.40%, 0.45%, 0.50%, 0.60%,0.70%, 0.80%, 0.90%, 1.00%, 1.10%, 1.20%, 1.30%, 1.40%, 1.50%, 1.60%,1.70%, 1.80%, 1.90% or 2.00%. Further, the fermented products obtainedusing the variant yeast strain has more glycerol than the fermentedproducts obtained using the ancestral yeast strain. For example, theratio of the glycerol content of a fermented product (e.g., wine)obtained with the variant yeast strain to the glycerol content of afermented product (e.g., wine) obtained with the ancestral yeast strain,is between about 1.25 and about 2.40 or at least about 1.25 1.30, 1.35,1.40, 1.45, 1.50, 1.55, 1.60, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00,2.10, 2.15, 2.20, 2.25, 2.30, 2.35 or 2.40. In some embodiment, the“variant” yeast strain, during an alcoholic fermentation, does notproduce an amount of acetate, acetaldehyde and acetoin (when compared tothe “ancestral yeast strain”) which can alter the organolepticproperties of the fermented product. For example, the content ofacetate, acetaldehyde or acetoin in the fermented product obtained byusing the variant yeast strain can be either equal to or less than thecorresponding content of acetate, acetaldehyde or acetoin in thefermented product obtained by using the ancestral yeast strain.Alternatively, the content of acetate, acetaldehyde or acetoin in thefermented product obtained by using the variant yeast strain can beaugmented when compared to the corresponding fermented product obtainedusing the ancestral yeast strain. In still some embodiments, the variantyeast strain can produce a greater amount of one or more compounds (suchas 2,3-butanediol), when compared to the ancestral yeast strain, whichdoes not impact the organoleptic properties of the fermented product. Insome embodiments, the variant yeast strains are not more resistant to anhyperosmotic shock caused by the salt than the ancestral yeast strain,but the variant yeast strains display better viability and a gain offitness (when compared to the ancestral yeast strain) under conditionsof hyperosmotic stress and carbon starvation.

One of the exemplary variant yeast strain of the present disclosure hasbeen deposited at Institut Pasteur, on Jan. 9, 2014, under accessionnumber CNCM I-4832. Another exemplary variant yeast strain of thepresent disclosure has been deposited at Institut Pasteur, on Oct. 18,2012 under accession number CNCM I-4684. A further exemplary variantyeast strain of the present disclosure has been deposited at InstitutPasteur, on Oct. 18, 2012 under accession number CNCM I-4685. In yetanother exemplary variant yeast strain of the present disclosure hasbeen deposited at Institut Pasteur, on Jan. 28, 2015 under accessionnumber CNCM I-4952.

The present disclosure also concerns the use of the variant yeast strainduring an alcoholic fermentation process in which it is warranted tolimit the alcohol content of the final fermented product. In the processfor making a fermented product having an alcoholic content, the variantyeast strain is placed in contact with a fermentable source of nutrientsand the fermentation is conducted in conditions allowing the completionof the alcoholic fermentation. The variant yeast strains are especiallyuseful in processes for making wines (e.g., red, white, rosé, sparklingor fortified wine). In such embodiment, the variant yeast strain isplaced into contact with a grape must and the fermentation is conductedin conditions allowing the completion of the alcoholic fermentation.Optionally, the variant yeast strain can be provided in a driedformulation and submitted to a rehydration step prior to thefermentation. In another embodiment, the variant yeast strain can beprovided in a liquid formulation and submitted to a dilution step and/ora thawing step prior to fermentation. In an embodiment, only the variantyeast strain is used to complete the alcoholic fermentation.Alternatively, the variant yeast strain can be admixed with other yeaststrain to ferment. In some embodiments, when the fermented product is awhite wine, the fermentation is conducted at a temperature below about25° C., usually at about between about 20° C. and about 24° C. In otherembodiments, when the fermented product is a red wine, the fermentationis conducted at a temperature equal to or higher than about 25° C., forexample, at a temperature between about 25° C. and about 30° C., and insome embodiments, at a temperature between about 25° C. and about 28° C.(e.g., 28° C. for example). In some variant yeast strains describedherein, a metabolic shift towards the production of glycerol has beenobserved when the yeasts are incubated at a temperature higher thanabout 24° C. In such variant yeasts strain, the maximal reduction inethanol production was observed at about 28° C. As such, some of thevariant yeasts strains described herein are especially suited forproviding a lower alcohol content in red wines. The resulting wines canoptionally be filtered and bottled, as it is currently done in the art.

The variant yeast strains can be used to ferment the must of differentgrape species (alone or in combination), such as Vitis vinifera, as wellas hybrid grape species combining one of more of V. labrusca, V.aestivalis, V. ruprestris, V. rotundifolia and V. riparia. The variantyeast strains can be used to ferment the must of different grapevarieties (alone or in combination) used to make red, white, rosé,sparkling or fortified wine. Grape varieties used to make red winesinclude, but are not limited to, Aghiorghitiko, Aglianico, Aleatico,Alicante Bouschet, Aramon, Baga, Barbera, Blaufrankisch, Cabernet Franc,Cabernet Sauvignon, Canaiolo, Carignan, Carmenere, Cinsaut, Dolcetto,Dornfelder, Elbling, Freisa, Gaglioppo, Gamay, Grenache/Garnacha,Grignolino, Malbec, Mavrud, Melnik, Merlot, Mondeuse (Refosco),Montepulciano, Nebbiolo, Negroamaro, Nero d'Avola, Nielluccio,Periquita, Petit and Gros Manseng, Petit Verdot, Petite Sirah,Sagrantino, Sangiovese, Saperavi, Saint Laurent, Syrah/Shiraz, Tannat,Tempranillo, Teroldego, Tinta Barroca, Tinto Cao, Touriga Francesa,Xinomavro and/or Zinfandel. Grape varieties used to make white winesinclude, but are not limited to, Airen, Albana, Albarino (Alvarinho),Aligote, Arneis, Bacchus, Bombino, Chardonnay, Chasselas, Chenin Blanc,Clairette, Ehrenfelser, Elbling, Ezerjo, Fernão Pires, Furmint,Garganega, Gewürztraminer, Grechetto, Greco, Grillo, Gruner Veltliner,Hárslevelú, Huxelrebe, lnzolia, lona, Jacquère, Kerner, Listan, Macabeo,Malvasia, Marsanne, Melon de Bourgogne, Optima, Palomino, Parelleda,Pedro Ximenez, Picpoul, Pinot Blanc, Pinot Gris/Grigio, Reichensteiner,Riesling, Rkatsiteli, Robola, Roditis, Sauvignon Blanc, Savagnin,Scheurebe, Semillon, Silvaner, Tocai Friulano, Torrontes, Trebbiano,Ugni-Blanc, Verdejo, Verdelho, Verdicchio, Vermentino, Vernaccia di SanGimignano, Viognier, Welschriesling and/or Xarel-lo.

Some of the advantages of using the variant yeast strain in processesfor making a fermented product (such as wine) include, but are notlimited to, the avoidance of using genetically-modified yeast strains,the avoidance of using mechanical de-alcoholisation procedures (e.g.,reverse osmosis, nano-filtration or distillation) and/or theapplicability to various grape varieties, irrelevant to the initialsugar content. As such, the process for obtaining a fermented producthaving alcohol (such as a wine) can exclude the use ofgenetically-modified yeast strain and/or the use of mechanicalde-alchololisation procedures.

The variant yeast strains are especially useful in processes for makingwines (e.g., red, white, rosé, sparkling or fortified wine). In suchembodiment, the variant yeast strain is placed into contact with a winemust and the fermentation is conducted in conditions allowing thecompletion of the alcoholic fermentation. Optionally, the variant yeaststrain can be submitted to a rehydration step prior to the fermentation.In an embodiment, only the variant yeast strain is used to complete thealcoholic fermentation. Alternatively, the variant yeast strain can beadmixed with other yeast strain to ferment. In some embodiments, whenthe fermented product is a white wine, the fermentation is conducted ata temperature below about 25° C., usually at about between about 20° C.and about 24° C. In other embodiments, when the fermented product is ared wine, the fermentation is conducted at a temperature equal to orhigher than about 25° C., for example, at a temperature between about25° C. and about 30° C., and in some embodiments, at a temperaturebetween about 25° C. and about 28° C. (e.g., 28° C. for example). Insome variant yeast strains described herein, a metabolic shift towardsthe production of glycerol has been observed when the yeasts areincubated at a temperature higher than about 24° C. In such variantyeasts strain, the maximal reduction in ethanol production was observedat about 28° C. As such, some of the variant yeasts strains describedherein are especially suited for providing a lower alcohol content inred wines. The resulting wines can optionally be filtered and bottled,as it is currently done in the art.

The variant yeast strains can also be used to ferment the cereal-derivedstarch (e.g. malted cereal) in brewed application for making beer.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—KCl-Based Adaptive Laboratory Evolution

Yeast Strain and Growth Conditions.

The wine yeast strain S. cerevisiae Lalvin EC1118® was used as theancestral strain. Prior to ALE, strains were propagated in rich YPDmedium (1% bacto yeast extract (DB), 2% bactopeptone (DB), 2% glucose;Legallais) or in synthetic SD medium (0.67% Difco yeast nitrogen basewithout amino acids (DB), 2% glucose) and maintained on YPD plates (2%agar) at 4° C. or stored at −80° C. in 20% glycerol.

KCl Resistance Assay.

EC1118 was grown in 60 mL YPD with KCl in concentrations varying from0.5 to 3M, at 28° C., under agitation. Optical density at 600 nm wasmeasured each 6 hours until 240 hours and growth was compared for thedifferent conditions.

Adaptive Laboratory Evolution (ALE).

Adaptive evolution was based on a long-term serial transfer procedureusing KCl as stress inducer. The strain EC1118 was cultured overnight at28° C. in 5 mL of YPD, and the resulting cell suspension was used toinoculate capped tubes (13 mL), each containing 5 mL medium with 1%bacto yeast extract, 2% bactopeptone, 14% glucose and 1.25M KCl(Sigma-Aldrich). Duplicate evolution experiments and also a controlwithout stress were performed. The cultures were incubated at 28° C.under agitation at 225 rpm. After 7 days, corresponding to about 5generations, the optical density of the culture at 600 nm (OD₆₀₀) wasmeasured and an aliquot was used to inoculate a fresh medium such thatthe OD₆₀₀ was 1. Such serial transfers were repeated for 450generations. Every 50 generations, 1 mL samples of the evolvingpopulation were taken and stored at −80° C. in 20% glycerol forsubsequent analysis.

After 7 days of culture, the cultures were inoculated in a YPD mediumcontaining 13.6% glucose and 1.30 M KCl. Then, each week, thesubcultures were inoculated in a medium in which the KCl concentrationwas increased by a further 0.1 M and the glucose concentration wasdecreased by a further 0.4%. This phase lasted between d₇ and d₄₉.Afterwards every 4 weeks, the KCl concentration was increased by 0.1 Mand the glucose was decreased by 0.4%. This phases lasted between d₄₉and d₇₂₈.

Wine Fermentation (Laboratory Scale).

Batch fermentation experiments were carried out in synthetic medium(MS), which mimics a standard grape juice. MS medium was prepared asdescribed by Bely et al. with the following modifications: 260 g/Lglucose, 210 mg/L available nitrogen, 7.5 mg/L ergosterol, 0.21 g/LTween® and 2.5 mg/L oleic acid (MS210 medium). Fermentations in grapemust were carried out in the same conditions, using Chardonnay-Coursan2011 previously flash pasteurized. The fermentations were performed in330 mL fermenters containing 300 mL medium, inoculated with 0.5×10⁶cells per mL and incubated at 28° C. with continuous stirring (350 rpm).To study the metabolic flexibility of the evolved and ancestral strains,different temperatures were used (16, 20, 24, 32 and 34° C.).Fermentation kinetics was monitored by calculation of the amount of CO₂released determined by weighing the fermenters manually. Allfermentation experiments were performed in triplicate. Extracellularmetabolites and volatile compounds were assayed at the end of thefermentation.

Wine Fermentation (Pilot Scale).

Pilot-scale fermentations were performed in 1 hL cylindricalstainless-steel tanks with Grenache variety grape must. This grape mustcontains 269 g/L sugars and 186 mg/L nitrogen and was flash pasteurizedand stored at 2° C. before fermentation. Grenache must was inoculated at25 g/hL with EC1118 and K300.1(b) active dry yeasts (Lallemand,Toulouse, France). CO₂ production was determined using a Brooks 5810 TRseries gas flowmeter (Brooks Instrument, PA, USA), as described byAguera and Sablayrolles. Fermentations were carried out under isothermalconditions at 28° C. Dissolved oxygen was added during fermentation tolimit the risk of stuck fermentation. A transfer of 4 mg/L, 7 mg/L and10 mg/L oxygen was performed when the CO₂ released reached 7.2 g/L, 13.5g/L and 45 g/L respectively. Nitrogen (72 mg/L) was added under the formof 15 g/hL DAP and 30 g/hL FermaidE at 45 g/L of CO₂ released.

Viability of Evolved Strains.

Ancestral and evolved cells were grown in 50 mL of YPGluKCl (1% bactoyeast extract, 2% bactopeptone, 8% glucose and 2.4 M KCl) inoculated at0.1 OD₆₀₀/mL from an overnight preculture in YPD. The size of the cellpopulation, extracellular metabolites and viability were followed for 7days. The assays were performed in triplicate. Viability was determinedusing a flow cytometer (Accuri, BD Biosciences) to count 20 000 cellsdiluted and washed in 300 μL 1×PBS (137 mmol/L NaCl (Sigma-Aldrich), 2.7mmol/L KCl, 100 mmol/L Na₂HPO₄ (Sigma), 2 mmol/L KH₂PO₄ (Sigma), pH 7.5)with 3 μL of propidium iodide (Calbiochem) previously diluted to 0.1mg/mL in sterile water.

Analytical Methods.

Cell densities were determined by measuring the OD₆₀₀ with a SecomamUVILine 9400 or by using a Coulter ZBI cell counter linked to a C56Channelyzer fitted with a probe with a 100 mm aperture (BeckmanCoulter). Dry weight was determined gravimetrically by filtering 10 mLof sample (pore size 0.45 μm, Millipore) and drying the sample for 24 hat 100° C. Extracellular glucose, glycerol, ethanol, pyruvate, succinateand acetate concentrations were determined by high-pressure liquidchromatography (HPLC), using an HPX-87H ion exclusion column (Bio-Rad).Volatile compounds (acetoin and 2,3-butanediol) were assayed by gaschromatography (GC). Acetoin and butanediol were extracted intochloroform according to the Hagenauer-Hener protocol with the followingmodifications: 1 mL of hexanol (Sigma) as an internal standard (1:1000v/v) in 10% ethanol (VWR) was added to 1 mL of sample. The organic phasewas dried and 1 μL was injected into a 30 m megabore column (DBWAX,JandW Scientific) on a GC apparatus HP 6890. The acetaldehydeconcentrations were determined enzymatically according to the Lundquistmethod. For pilot-scale experiments, glucose and fructose concentrationswere determined enzymatically. The ethanol concentration was determinedby measuring density, the volatile acidity by the bromophenol bluemethod, the SO₂ concentration by iodometry and total acidity bytitration. The osmolality was measured using a Vapro 5520 device(Wescor) with a sample volume of 10 μL.

Adaptive evolution under hyperosmotic KCl-medium and isolation ofhigh-glycerol-producing evolved strains. To evolve strain EC1118, batchcultures in YPD 8% glucose with a gradual increase of osmotic stresswere performed. KCl stress was chosen because it generates osmotic andsalt stress but unlike NaCl does not cause cation toxicity. A high sugarconcentration (8%) was used to maintain good fermentative performancesof the evolved strain in rich sugar medium. In preliminary experiments,the effect of various KCl concentrations was tested on EC1118 growth andit was found that the addition of 1.25 M KCl on YPD 8% glucose reducedthe growth of EC1118 four times (data not shown). The adaptivelaboratory evolution (ALE) experiments were started in YPD 8% glucosecontaining 1.25 M KCl. The osmolality of this medium is 2 105 mmol/kg,compared to 480 mmol/kg for YPD 8% glucose. The concentrations of KClwere progressively increased up to 2.4 M, corresponding to an osmolalityof 3730 mmol/kg and maintained at that level thereafter. Duplicate ALEexperiments were performed for each condition and one control ALEexperiment, without osmotic stress, was done.

Samples collected after 100, 200, 300 and 400 generations were firstanalysed to monitor the dynamics of each evolution experiment. Yeastcells were plated on YPD and the populations obtained were characterizedduring fermentation of the synthetic must MS210 at 28° C. The glycerolconcentration in the growth medium was measured at the end of thefermentation as a first indicator of the success or failure of theadaptation. Adaptation on KCl medium generated evolved populations withincreased glycerol production during wine fermentation (FIG. 1) whereasno increase of glycerol was observed in the control experiment(evolution of EC1118 without stress). A similar increase in glycerolproduction was observed in the two parallel KCl experiments (a) and (b).In fermentations with both (a) and (b) lineages, the concentration ofglycerol produced by fermentation reached 12 g/L for evolved populationsat 200 generations; the value for the ancestral EC1118 was 8.5 g/L. TheKCl-ALE experiment was pursued for 450 generations (total duration ofalmost 2 years), but only little variation in glycerol production wasobserved after 200 generations (data not shown).

First Characterization of the Evolved Strains During Wine Fermentation.

After several generations, due to the natural accumulation of mutations,a non-homogeneous population of yeasts should be present in samplesobtained from ALE experiments. Yeast populations sampled after differenttimes of the KCl-ALE experiment were subcultured on YPD. Thesesubclones, hereafter called evolved strains, were characterized duringwine fermentation on MS210 medium (FIG. 1). All the evolved strainsobtained after 200 generations produced more glycerol than the ancestralstrain; glycerol production remained stable after 200 generations.Consistent with the re-routing of carbons and NADH oxidation resultingfrom increased glycerol production, all evolved strains showed a reducedethanol yield. The ethanol yield was between 0.440 and 0.450 for theevolved strains and 0.464 for the reference ancestral strain (FIGS. 1 Aand B). The evolved mutants showed reduced sugar consumption (FIGS. 1 Cand D). Thus, there was a correlation between high glycerol yield,reduced ethanol yield and diminution of fermentative properties. Adetailed study of six KCl-evolved strains from populations isolatedafter 200, 250 and 300 generations, including three from lineage (a):K200.1(a), K250.1(a), K300.2(a) and three from lineage (b): K200.1(b),K250.3(b), K300.1(b) was undertaken. Yeast strain K300.1(b) (also namedLowa3 herein) was registered under CNCM I-4684 as a biological depositin the Collection National de Cultures de Microorganismes (CNCM) of theInstitut Pasteur on Oct. 18, 2012. Yeast strain K250.3(b) (also namedLowa2 herein) was registered under CNCM I-4685 as a biological depositin the Collection National de Cultures de Microorganismes (CNCM) of theInstitut Pasteur on Oct. 18, 2012.

High-Glycerol-Producing Strains Survive Better in Conditions of OsmoticStress and Carbon Restriction.

The resistance of the evolved strains to hyperosmotic stress wasassessed by growth on KCl, NaCl or sorbitol SD plates. In theseconditions, no significant differences in growth were observed betweenEC1118 and the evolved strains (FIG. 2): under the conditions of the ALEexperiment (YPD 80 g/L glucose, 2.4 M KCl), the specific growth rate andmaximal cell number reached by the evolved strains were similar to thoseof the ancestral strain. Therefore, yeast cells that evolved in theseconditions did not display growth adaptation to osmotic stress. It wasthen examined whether other components of fitness, such as viability,had been improved during the evolution experiment. Cell viability wasmonitored during culture involving a 7-day transfer cycle in theconditions of the evolution experiment (YPD 80 g/L glucose, 2.4 M KCl).After complete glucose exhaustion (about 4 days), the evolved mutantssurvived better than the ancestral strain. After 7 days (correspondingto the time of transfer to fresh medium during the evolutionexperiment), almost all EC1118 cells had died, whereas the number ofviable cells of the evolved mutants was considerably higher (FIG. 2).The viability of the evolved mutants at 7 days correlated with glycerolproduction at the same time-point (data not shown). Therefore, the mainadaptation to the selective pressure put on yeast cells during theadaptive evolution experiment appeared to be improved survival inconditions of salt stress and carbon restriction.

Characterization of the Selected KCl-Evolved Strains During WineFermentation.

The characteristics of the six selected KCl-evolved strains and theancestral strain were studied in detail during wine fermentation inanaerobic batch cultures on MS medium. All the strains were able tocomplete the fermentation, although the duration of the fermentationdiffered between the evolved strains (Table 1). Two evolved strains,K200.1(b) and K300.1(b), consumed all the sugar in less than two weeks,like the ancestral strain, whereas one month or more was required forthe four other evolved strains (sugar was completely exhausted onlyafter 40 days by K250.1(a) and K300.2(a)).

The fermentation rate of two evolved strains having distinctfermentation capacity, K300.2(a) and K300.1(b), is shown in FIG. 3A. Theevolved strains exhibited an overall decrease of fermentationperformance in comparison to the ancestral strain, which is consistentwith the reduced sugar consumption observed before, but werenevertheless able to complete the fermentation. Final cell populationwas the same between ancestral and these two evolved strains despitethat K300.2(a) showed a slower growth than K300.1(b) (FIG. 3B).

The concentration of the most abundant by-products was determined after30 days of fermentation (Table 1). Carbon and redox balances were closeto 100% for all strains. All evolved strains produced glycerol atconcentrations 48 to 67% higher than that produced by EC1118, and theethanol content in the synthetic wines was 0.45 to 0.80% (v/v) lower.The evolved strains also produced greater amounts of succinate,2,3-butanediol and acetaldehyde than the ancestral strain. Succinateproduction by K200.1(a) and K300.2(a) was 22% and 88.9% higher, thanthat by EC1118; the production of acetaldehyde by K200.1(a) andK300.2(a) was 45.5% to 181.8% higher, respectively, and that of2,3-butanediol by 93% to 255.6% higher. The concentration of thesecompounds was also increased in strains overexpressing GPD1 coding forthe glycerol 3-P dehydrogenase, in which the carbon flux is redirectedtowards glycerol formation at the expense of ethanol (Michnick et al.,Remize et al., Cambon et al.). By contrast, unlike previously describedengineered strains, no significant changes in the production of acetateand acetoin by the evolved strains was observed.

Although similar phenotypes were observed for the two replicates,lineage (b) was characterized by slightly greater glycerol productionand better fermentative performances. K300.1(b) was the most promisingevolved strain obtained in terms of fermentation capacity and productionof glycerol, succinate, 2,3-butanediol and ethanol.

Metabolic Properties of the Evolved Strain K300.1(b) at VariousTemperatures on Synthetic and Natural Grape Musts.

Wine can be produced in a large range of fermentation temperatures,usually from 16° C. (for white wines) to 28° C. and more (for redwines). The metabolic properties of the ancestral strain and the evolvedstrain K300.1(b) were compared over a wide range of temperatures (16,20, 24, 28, 32 and 34° C.) in MS210 medium containing 260 g/L sugars.For temperatures between 16 and 28° C., both strains consumed all ormost of the sugar, while for the two highest temperatures, a residualsugar concentration of 43 and 53 g/L for EC1118 and 47 and 59 g/L forK300.1(b) was observed at 32 and 34° C. respectively.

The yields of by-products were determined after 30 days of fermentation(FIG. 4). At all temperatures, K300.1(b) was clearly differentiated fromEC1118 on the basis of high glycerol, high succinate and low ethanolyields. The yields of glycerol and succinate increased with increasingtemperature, whereas the ethanol yield decreased. The differencesbetween temperatures were larger for K300.1(b) than for EC1118, inparticular for the three highest temperatures. The ethanol content wasreduced by 0.14% (v/v), 0.18% (v/v) and 0.24% (v/v) at 16° C., 20° C.and 24° C. and by 0.61% (v/v), 0.80% (v/v), 0.87% (v/v) at 28° C., 32°C. and 34° C. respectively with the evolved strain compared to EC1118.Therefore, a metabolic shift was observed between 24° C. and 28° C. Toexamine whether a similar behavior can be observed on natural must,fermentation was carried out in Chardonnay-Coursan, under similarconditions, at 24° C. and 28° C. Under these conditions, the ethanollevel was reduced by 0.12% (v/v) at 24° C. and 0.42% (v/v) at 28° C.,confirming the results obtained in synthetic must. These resultshighlight a more flexible metabolism in the evolved strain regardingtemperature, with the reduction of ethanol yield maximized at atemperature of 28° C. and above.

TABLE 1 Metabolites, carbon and redox balances, and markers offermentation for EC1118 and evolved strains measured after 30 days offermentation on MS210, 260 g/L glucose, 28° C. Main compounds (g/L)EC1118 K200.1(a) K250.1(a) K300.2(a) K200.1(b) K250.3(b) K300.1(b)consummed glucose 269.9 ± 0.1  258.5 ± 1.4  250.7 ± 4.5  251.3 ± 5.0 260.0 ± 0.1  258.6 ± 1.5  259.8 ± 0.3  CO₂ 117 ± 0  112 ± 1  110 ± 1 109 ± 1  112 ± 0  112 ± 2  110 ± 1  biomass (80%)* 4.0 ± 0.1 3.8 ± 0.14.0 ± 0.2 3.7 ± 0.2 3.6 ± 0.2 3.9 ± 0.0 4.0 ± 0.1 ethanol 120.6 ± 0.6 116.3 ± 1.3  112.9 ± 1.5  113.2 ± 1.0  116.3 ± 0.7  115.5 ± 1.6  114.0 ±0.8  glycerol 8.5 ± 0.1 13.2 ± 0.1   12.7 ± 0.1  12.6 ± 0.2  13.3 ± 0.5 13.9 ± 0.2  14.2 ± 0.3  succinate 0.9 ± 0.0 1.5 ± 0.1  1.5 ± 0.1  1.1 ±0.1 1.5 ± 0.1 1.5 ± 0.1 1.7 ± 0.1 pyruvate 0.22 ± 0.03 0.20 ± 0.00 0.20± 0.01 0.19 ± 0.00 0.20 ± 0.01 0.19 ± 0.01 0.19 ± 0.02 acetate 0.9 ± 0.20.7 ± 0.0 0.9 ± 0.0 1.1 ± 0.0 1.0 ± 0.1 1.0 ± 0.2 1.0 ± 0.1 acetaldehyde0.011 ± 0.001 0.016 ± 0.001 0.023 ± 0.002 0.031 ± 0.007 0.025 ± 0.0050.031 ± 0.010 0.029 ± 0.001 acetoin nd nd nd nd nd nd nd 2,3-butanediol0.45 ± 0.02 1.00 ± 0.09 0.89 ± 0.30 1.08 ± 0.14 0.87 ± 0.11 1.10 ± 0.181.60 ± 0.06 Carbon balance^(a) (%) 97.6 ± 0.6  96.9 ± 0.8  97.2 ± 2.5 97.0 ± 2.7  96.4 ± 0.8  96.7 ± 0.8  95.9 ± 0.5  Redox balance^(b) (%)97.4 ± 0.6  97.3 ± 0.7  97.5 ± 2.1  97.3 ± 2.1  96.6 ± 0.8  97.1 ± 1.7 96.4 ± 0.6  YEtOH 0.464 ± 0.002 0.450 ± 0.004 0.450 ± 0.010 0.450 ±0.010 0.447 ± 0.003 0.447 ± 0.009 0.440 ± 0.003 Yglycerol 0.033 ± 0.0000.051 ± 0.000 0.051 ± 0.001 0.050 ± 0.002 0.051 ± 0.002 0.054 ± 0.0010.054 ± 0.001 Yglycerol/YEtOH (%) 7.05 ± 0.07 11.32 ± 0.09  11.27 ±0.11  10.09 ± 0.23  11.46 ± 0.40  12.01 ± 0.26  12.38 ± 0.27  ethanol**(% (v/v)) 15.29 ± 0.08  14.82 ± 0.13  14.84 ± 0.32  14.84 ± 0.34  14.74± 0.09  14.72 ± 0.29  14.50 ± 0.09  glucose (g) for 1% (v/v) ethanol17.00 ± 0.07  17.53 ± 0.09  17.52 ± 0.11  17.52 ± 0.23  17.64 ± 0.40 17.67 ± 0.26  17.97 ± 0.27  residual sugar^($) (g/L) 0.1 ± 0.1 20.2 ±1.4  58.7 ± 4.5  30.4 ± 5.0  0.1 ± 0.1 22.5 ± 1.5  0.2 ± 0.3 residualsugar^($$) (g/L) 0.1 ± 0.1 1.5 ± 1.4 9.3 ± 4.5 8.7 ± 5.0 0.0 ± 0.1 1.4 ±1.5 0.1 ± 0.3 ^(a)Carbon balance represents the ratio between carbonmoles of fermentation by-products and carbon moles of glucose. ^(b)Redoxbalance represents the ratio between the reductance degree offermentation by-products and the reductance degree of glucose. Bothbalances are expressed in percentage. nd: not detected (<10 mg/L)*Biomass measured at 80% of fermentation advancement **Potential ethanol^($)Measured after 15 days ^($$)Measured after 30 days

Pilot-Scale Assessment of the Evolved Strain K300.1(b).

To validate the results obtained at laboratory scale, the behavior andthe metabolic properties of K300.1(b) and EC1118 were compared duringpilot scale fermentation, using a Grenache grape must, at 28° C. (Table2).

TABLE 2 Characteristics of wines obtained by fermentation of Grenachemust with EC1118 and K300.1(b) at pilot scale (1 hL). ASV was determinedby distillation and electronic densitometry. EC1118 K300.1(b) residualsugars (g/L) 0.2 0.5 volatil acidity (g/L) 0.50 0.36 total acidity (g/L)4.10 4.40 ASV % v/v) 16.26 15.80 pH 3.60 3.57 free SO₂ (g/L) 0.07 0.07total SO₂ (g/L) 0.035 0.039 acetaldehyde (g/L) 0.021 0.030 malate (g/L)1.35 1.38 succinate (g/L) 1.04 1.43 acetate (g/L) 0.47 0.31 glycerol(g/L) 10.8 14.2

To be as close as possible to industrial conditions, the strainsK300.1(b) and EC1118 were used in the form of active dry yeast andinoculated after a standard rehydration procedure. To avoid stuckfermentation, oxygen and nitrogen were added during fermentation (FIG.5). The evolved strain had a fermentation rate slightly lower thanEC1118, but was able to complete the fermentation, despite the highsugar concentration (270 g/L). The evolved strain produced more glycerol(14.2 g/L versus 10.8 g/L) and succinate than EC1118, and less ethanol,resulting in a reduction of 0.46% (v/v) of the ethanol content. Theseresults are in agreement with those obtained at laboratory scale. Theproduction of acetic acid and volatile acidity by the evolved strain wasclearly lower than for EC1118. The production of acetic acid for bothstrains was much lower than on synthetic must, which is in agreementwith previous observations. In summary, the results obtained in grapemust at pilot-scale confirm the metabolic reprogramming of the evolvedstrain, and the analysis of the wine obtained did not reveal adverseside effects.

In this study, adaptive laboratory evolution (ALE) was used to developlow-alcohol wine yeasts by redirecting the metabolism of strain EC1118towards glycerol. Yeast cultures were serially transferred inhyperosmotic conditions during 450 generations using KCl as osmo- andsalt-stress salt. The stress imposed was severe, from osmolalities of 2105 to 3 730 mmol/kg. These levels of stress are above those generallyused in laboratory conditions to study responses to osmotic stress (20g/L glucose, 1.2 M NaCl, corresponding to an osmolality of 2 070mmol/kg). The KCl stress generated strains in which the carbon flux wasre-directed towards glycerol. In another experiment, sorbitol was usedas an osmotic salt (from 1.5 to 2.4 g/L corresponding to 1 480 to 2 105mmol/kg), but these conditions failed to generate strains with increasedglycerol production (data not shown). This clear difference in effectmay be a consequence of the different natures of the stress salt (saltversus osmotic stress), and/or the higher level of stress in the KCl-ALEexperiment than the sorbitol-ALE experiment. The evolved strainsobtained from the KCl-ALE experiment were not more resistant than theancestral strain to osmotic or salt stress, but showed a gain of fitnessdue to a better viability under conditions of salt stress and carbonstarvation, the conditions in which cells were transferred to a freshmedium. No increase in glycerol production was observed in the ALEcontrol experiment with EC1118 without KCl stress (data not shown).Therefore, it is likely that the redirection of carbon fluxes towardsglycerol was driven by the combination of high KCl concentration andcarbon starvation stresses.

The link between survival and glycerol is intriguing. Usually, cells dieafter the culture enters the stationary phase, when one or all of thenutrients are missing. However, if the only nutrient missing is thecarbon source, cells survive longer. Without wishing to be bound totheory, it is stipulated that, under carbon limitation, nutrient sensingdepends on Sch9, Tor, and Ras proteins that are activated and convergeon the protein kinase Rim15; Rim15 regulates the transcription factorsMsn4/Msn2 and Gis1, involved in cellular protection and longevity, alsocalled chronological life span (CLS). Recent work indicates thatglycerol production is required for CLS regulation), and variousdistinct mechanisms have been suggested. Unlike glucose and ethanol,glycerol does not inhibit the transactivation of Msn2/Msn4 and Gis1,which play important roles in general stress resistance and longevity.However, glycerol production may affect aging through the modulation ofthe intracellular redox balance, because its production contributes tothe maintenance of the NAD⁺/NADH ratio. Overexpression of themalate-aspartate NADH shuttle was also demonstrated to extend the CLS.Also, high osmolarity has been postulated to extend the life span byactivating Hog1, leading to an increase in the biosynthesis of glycerolfrom glycolytic intermediates. Links between aging and redox metabolismduring wine fermentation have also been highlighted.

The detailed characterization of the KCl-evolved mutants during winefermentation revealed that the evolved strains had undergone substantialchanges to their central carbon metabolism: carbons in these strains aremainly re-routed towards glycerol, succinate and 2,3-butanediol at theexpense of ethanol. The absence of stress resistance phenotype and theimproved fitness under carbon restricted and stress conditions suggestthat the primary target of evolution is not the HOG pathway. The originof the observed phenotype might rely on indirect mutations disturbingthe redox balance, causing a redirection of carbon flux. Other factorssuch as a lower glucose uptake rate might also play a role in thephenotype. Indeed, the net flux through the TCA cycle increasedsignificantly with decreasing glucose uptake, which is reminiscent ofthe increased succinate production and lower fermentation rate in theevolved strains. On the other hand, it was previously shown thatglycerol production is less dependent on rate of glucose uptake and moreinfluenced by environmental conditions. Other studies using genome-wideapproaches may be required to elucidate the underlying mechanisms.

As observed previously in engineered strains overexpressing GPD1(Michnick et al., Remize et al., Cambon et al.), increased glycerolproduction is associated with a reduction of ethanol synthesis due tolower carbon availability and NADH shortage, and this is accompanied byperturbations at the acetaldehyde and pyruvate nodes. For example,strains overexpressing GPD1, and producing large amounts of glycerol butlow ethanol levels, accumulate succinate and 2,3-butanediol but alsoundesirable compounds including acetaldehyde, acetate and acetoin(Remize et al., Cambon et al.). The evolved strain described herein didnot accumulate high levels of these compounds, possibly due, forexample, to the smaller increase in glycerol production than in theengineered strains, and/or to a different metabolic strategy. In yeast,acetoin is reduced to 2,3-butanediol by the 2,3-butanedioldehydrogenase. It was previously showed that the balance between acetoinand 2,3-butanediol in the engineered strains can be influenced by theamounts of glycerol produced. In strains producing high glycerol levels,acetoin accumulated because of the limited capacity of the 2,3butanediol dehydrogenase and the decreased availability of NADH, as thiscofactor is mainlyre-oxidized through glycerol synthesis. In a previousstudy (Michnick et al.), it was found that strains overproducingglycerol at moderate levels (such as W18GPD1 or W6GPD1), comparable tothe evolved mutants characterized herein, did not accumulate acetoin. Asthe evolved strains, these strains also accumulated acetaldehyde at lowlevels, which can be explained by a limitation of the alcoholdehydrogenase. These levels remain in the range of usual concentrationsin wines and are unlikely to cause a sensory problem. In contrast, thereduced accumulation of acetate by the evolved mutants is surprisingbecause there was acetate accumulation in all cases, independent of thelevel of glycerol accumulated by the GPD1 strains (Blomberg et al.).This suggests that the modifications of the metabolic network in theevolved mutants differ from those in the genetically engineered strains.Without wishing to be bound to theory, another major difference involvesthe compromised fermentation performances of the evolved strains,suggesting that the mutations responsible for the re-routing ofmetabolism in these strains also negatively affect the glycolytic rate.This finding contrasts with the improved fermentation performances ofGPD1 strains during the stationary phase of wine fermentation.

It is thus hypothesized that adaptive evolution resulted in theutilization of routes different to those operating in rationallyengineered strains. The present disclosure provides the firstdescription of a non-GMO strategy allowing a substantial increase inglycerol production and decrease in the alcohol yield of a commercialwine yeast strain. A much higher diversion of carbon was obtained whencompare to previous attempts to divert carbons towards the pentosephosphate pathway or towards glycerol by adaptive evolution usingsulfites (Kutyna et al.). Consequently, the reduction in the ethanolcontent in of wine produced with our strains was at least 0.5% (v/v)from 260 g/L sugars. Despite the lower fermentation performances of theevolved strains, evolved isolates with only slightly affectedfermentation kinetics were selected. A first assessment of the potentialvalue of the evolved strain K300.1(b) for winemaking revealed similarcharacteristics in synthetic and natural grape musts, except thatacetate production was reduced in wines obtained from grape musts.Interestingly, evolved strain K300.1(b) has a higher metabolicflexibility than the ancestral strain with respect to temperature, withmetabolic differences between the two strains being greatest attemperatures higher than 24° C. This suggests that the evolved strainmight be particularly useful for the production of red wines, which areusually produced in a temperature range of 25−30° C. and are mostaffected by excessive alcohol levels.

The present example demonstrates that the adaptive evolution strategyused herein is a valuable alternative to rational engineering for thegeneration of non-GMO, low-ethanol producing yeast. Although thediversion of carbon flux obtained is not as high as that achieved bygenetic engineering, a reduction of the alcohol content of wine by 0.5to 1% (v/v) offers interesting perspectives. A preliminary wine tastingby a panel of seven wine experts did not revealed any defect of thewines produced at pilot scale, confirming the good overall attributes ofthe evolved strains reported in this study.

Example II—Second Generation of Low-Ethanol Producing Yeasts Obtained byBreeding

The S. cerevisiae strain K300.1(b) (obtained and characterized inExample I and renamed Lowa3 in Example II) was cultured (2 days at 28°C.) in a presporulation GNA medium (BactoYeast extract 1%, BactoPeptone2%, glucose 20%, agar 2%). The Lowa3 strain was then transferred in asporulation spoMA medium (BactoYeastExtract 0.1%, glucose 0.05%,potassium acetate 1%, adenine 0.002%) and cultured between 3 to 15 daysat room temperaure (about 20° C.) to produce asci. The asci obtainedwere isolated and incubated with the digestive juice of the snail HelixPomatia (1/6 dilution) for 20 to 60 min at 28° C. The asci weredissected with a dissection microscope (Singer MSM300) to isolate thespores. With this method, about 700 spores were isolated.

Since the S. cerevisiae strain EC1118 and the Lowa3 strain areheterozygotes for the HO gene, it is expected that half of the progenywill be haploid, whereas the other half of the progeny will be diploid.As such, a PCR selection was undertaken to select the haploid progeny(e.g., HO^(−/−)) of the Lowa3 strain. The PCR was performed on a part ofa colony the diluted in 50 μL of sterile water and heated for 10 min at95° C. to liberate the DNA. Five (5) μL of the aqueous DNA mixture wasadmixed to 10× Taq buffer (with (NH₄)₂SO₄; 2.5 μL), 25 mM MgCl₂ (2.5μL), 10 mM dNTPs (0.5 μL), the MAT-F primer (1 μL), the MATa-R orMATalpha-R primer (1 μL), Taq polymerase (Fermentas, 0.25 μL) and waterto obtain a final volume of 25 μL. The following oligonucleotides wereused to distinguish between the MATa and MATalpha genes: Mat F(5′-AGTCACATCAAGATCGTTTATGG-3′) (SEQ ID NO: 1), Mat a-R(5′-ACTCCACTTCAAGTAAGAGTTTG-3′, generating a 504 bp amplicon of the MATagene) (SEQ ID NO: 2) and Mat alpha-R (5′-GCACGGAATATGGGACTACTTCG-3′,generating a 404 bp amplicon of the MATalpha gene) (SEQ ID NO: 3). ThePCR was conducted during 30 cycles using a denaturation temperature of94° C. (for 1 min), an hybridization temperature of 55° C. (for 1 min)and an elongation temperature of 72° C. (for 1 min).

Using the PCR selection, 156 haploid spores of the Lowa3 strain wereobtained. The phenotype of the haploid spores was further characterizedduring wine fermentation. Several wine fermentations were conducted in330 mL fermenters containing 300 mL synthetic must (MS425, 260 g/Lglucose) under agitation. After 15 and 30 days of fermentation at 28°C., a sample of the supernatant was obtained to determine the glycerolconcentration. The different haploid strains were classified in functionof their glycerol production (which is inversely proportional to theirethanol production). The best strain of the MATa mating type, named5074, produced 20.9 g/L glycerol. The best strain of the MATalpha matingtype, named 5049, produced 16.2 g/L glycerol. Strains 5074 and 5049 wereselected for further breeding.

Both strains were cultured to be in their growth phase and werecontacted in a YPD medium (BactoYeast extract 1%, BactoPeptone 2%,glucose 2%, agar 2%). A first hybrid was obtained, named VT1 (H1generation hybrid). The diploid nature of the VT1 hybrid was confirmedby the absence of breeding when it was placed in contact with a strainof the MATa mating type and with a strain of the MATalpha mating type.

Spores of the hybrid strain VT1 were generated with the medium GNA andspoMA as described above. A stable haploid spore of the hybrid VT1strain, named MP120-A4 was selected based on its MATalpha mating type.The strain MP120-A4 was bred with the strain 5074 to obtain the strainMP112-A5 (H2 generation hybrid). Strain MP112-A5 (also referred as H2)was registered under CNCM I-4832 as a biological deposit in theCollection National de Cultures de Microorganismes (CNCM) of theInstitut Pasteur on Jan. 9, 2014.

The various strains obtained were further characterized using alaboratory scale or pilot scale wine fermentation, as described inExample I, and following the kinetics of each fermentations.

Fermentation Trial N1.

A synthetic must was used and has an initial concentration in sugar of235 g/L (117.5 g/L of glucose and 117.5 g/L of fructose). The initialconcentration in available nitrogen in this synthetic must was 300 mg/L.All the fermentations were conducted in isotherm conditions at 28° C.,in 1.1 L-containing fermenters.

As shown on FIG. 6, 112-A5 (H2) was compared with EC1118 to ferment thesame must with a 14% v/v potential alcohol. As previously described, thefermentation kinetic profiles are very different on the exponentialphase with a better fermentation rate of EC1118. The evolved yeaststrain (112-A5) had a long stationary phase and completed thefermentation much later than EC1118. However, the fermentation went todryness, meaning there is no residual sugars. This is confirmed by theanalysis in Table 4. The use of 112-A5 allowed to ferment with asignificant lower final ethanol level. Another difference is observed onthe final acetate content which is significantly lower with 112-A5.Further differences between the fermentations obtained using the EC1118or the 112-A5 strains are presented at Table 4.

TABLE 4 Analysis of various constituents of the fermented wine obtainedat fermentation trial N1 using the EC1118 or the 112-A5 strains. Theglycerol content was not determined. Acetate Residual Sugar/Ethanol ASVcontent sugars yield (% v/v) (g/L) (g/L) EC1118 16.67 14.09 0.66 <0.4112-A5 17.5 13.45 0.5 <0.4

Fermentation Trial N2.

A synthetic must was used and has an initial concentration in sugar of260 g/L (130 g/L of glucose and 130 g/L of fructose). The initialconcentration in available nitrogen in this synthetic must was 300 mg/L.All the fermentations were conducted in isotherm conditions at 28° C.,in 1.1 L-containing fermenters.

As shown on FIG. 7, 112-A5 (H2) was compared with EC1118 to ferment thesame must with a 15.6% v/v potential alcohol. As previously described,the fermentation kinetic profiles are very different on the exponentialphase with a better fermentation rate of EC1118. The evolved yeaststrain (112-A5) had a long stationary phase and completed thefermentation much later than EC1118. However, the fermentation went todryness, meaning there is no residual sugars. This is confirmed by theanalysis in Table 5. The use of 112-A5 allowed to ferment with asignificant lower final ethanol level. Another difference is observed onthe final acetate content which is significantly lower with 112-A5.Further differences between the fermentations using the EC1118 or the112-A5 strains are presented at Table 5.

TABLE 5 Analysis of various constituents of the fermented wine obtainedin fermentation trial N2 using the EC1118 or the 112-A5 strains. Theglycerol content was not determined. Acetate Residual Sugar/Ethanol ASVcontent sugars yield (% v/v) (g/L) (g/L) EC1118 16.7 15.56 0.68 0.4 H217.6 14.75 0.51 1.0

Fermentation Trial N3.

A Syrah variety grape must was used in this pilot scale fermentationtrial. The must was flash pasteurized and stored at 2° C. prior tofermentation. Prior to fermentation, the Syrah must had the followingcharacteristics: 255 g/L of total sugars, 3.50 g/L H₂SO₄ of totalacidity, pH=3.63, 138 mg/L of available nitrogen and a turbidity of 77NTU. Prior to fermentation, and as indicated in Example I, the yeastswere rehydrated. Further, during fermentation, the must was supplementedwith oxygen and nitrogen (as indicated in Example I). All thefermentations were conducted in isotherm conditions at 28° C., in 1hL-containing fermenters. Afterwards, the wines were bottled.

FIG. 8 shows the fermentative kinetics of EC1118, K300.1(b) (Lowa3) and112-A5 (H2) in real grapes, in enological conditions. The profiles aredifferent but the performances on the total fermentation duration quitesimilar, showing a delay of only 20 h for the fermentation with 112-A5.This confirms that 112-A5 is suitable to ferment high sugar grapes tilldryness, without stuck or sluggish fermentations. This is confirmed bythe analysis of the classical enological parameters reported in table 6Aand 6B. The final ethanol level shows a decrease of more than 1% for thewine fermented with 112-A5, with a higher glycerol production and a verylow acetate production (not detected). Further differences between thefermentations using the EC1118 or the 112-A5 strains are presented atTables 6.

TABLE 6A Kinetics parameters of the wine fermentation using the EC1118or the 112-A5 strains in fermentation trial N3. Vmax Time of Latency(g/L/h) Residual fermentation period after sugars trial (h) addition(g/L) (h) EC1118 11 2.13 0.3 130 112-A5 1 1.47 0.4 150

TABLE 6B Analysis of various constituents of the fermented wine obtainedin fermentation trial N3 using the EC1118, the K300.1(b) or the 112-A5(H2) strains. Measurements were done in triplicates. Main compounds(g/L) EC1118 K300.1(b) H2 consumed sugar 254.6 ± 0.1  254.5 ± 0.0  254.7± 0.2  ethanol 118.4 ± 1.2  113.6 ± 0.9  107.8 ± 0.8  glycerol 10.8 ±0.4  14.1 ± 0.4  17.9 ± 0.8  succinate 1.3 ± 0.1 1.8 ± 0.1 1.5 ± 0.1pyruvate 0.13 ± 0.01 0.16 ± 0.01 0.15 ± 0.01 acetate 0.5 ± 0.1 0.1 ± 0.0nd acetaldehyde 0.016 ± 0.008 0.021 ± 0.001 0.020 ± 0.006 acetoin nd nd0.024 ± 0.005 2,3-butanediol 1.11 ± 0.18 1.98 ± 0.38 3.93 ± 0.30 YEtOH0.465 ± 0.005 0.446 ± 0.003 0.423 ± 0.003 Yglycerol 0.042 ± 0.002 0.055± 0.000 0.070 ± 0.003 Yglycerol/YEtOH (%) 9.09 ± 0.34 12.37 ± 0.05 16.57 ± 0.84  ethanol (%(v/v)) 15.01 ± 0.15  14.40 ± 0.11  13.67 ± 0.10 glucose (g) for 16.99 ± 0.07  17.71 ± 0.07  18.66 ± 0.08  1% (v/v)ethanol nd: not detected (<10 mg/mL)

While the invention has been described in connection with specificembodiments thereof, it will be understood that the scope of the claimsshould not be limited by the preferred embodiments set forth in theexamples, but should be given the broadest interpretation consistentwith the description as a whole.

REFERENCES

-   Aguera E, Sablayrolles J M. Pilot scale vinifications (100 L). III    Controlled fermentation. Wine Internet Tech. J. 8.-   Bely M, Sablayrolles J M, Barre P. 1990. Automatic detection of    assimilable nitrogen deficiencies during alcoholic fermentation in    oenological conditions. J. Ferment. Bioeng. 70:246-252.-   Blomberg A, Adler L. 1992. Physiology of osmotolerance in fungi.    Adv. Microb. Physiol. 33:145-212.-   Cambon B, Monteil V, Remize F, Camarasa C, Dequin S. 2006. Effects    of GPD1 overexpression in Saccharomyces cerevisiae commercial wine    yeast strains lacking ALD6 genes. Appl. Environ. Microbiol.    72:4688-4694.-   Hagenauer-Hener U, Henn D, Fettmar F, Mosandl A, Schmitt A. 1990.    2,3 Butanediol-Direkte Bestimmung der Stereoisomeren im Wein. Dtsch    Leb. Rundsch 273-276.-   Kutyna D R, Varela C, Stanley G A, Borneman A R, Henschke P A,    Chambers P J. 2012. Adaptive evolution of Saccharomyces cerevisiae    to generate strains with enhanced glycerol production. Appl.    Microbiol. Biotechnol. 93:1175-1184.-   Lundquist F. Acetaldehyd: Bestimmung mit Aldehyd-dehydrogenase.    Methods of enzymatic analysis. Methods Enzym. Anal.-   Michnick S, Roustan J L, Remize F, Barre P, Dequin S. 1997.    Modulation of glycerol and ethanol yields during alcoholic    fermentation in Saccharomyces cerevisiae strains overexpressed or    disrupted for GPD1 encoding glycerol 3-phosphate dehydrogenase.    Yeast Chichester Engl. 13:783-793.-   Remize F, Roustan J L, Sablayrolles J M, Barre P, Dequin D. 1999.    Glycerol overproduction by engineered Saccharomyces cerevisiae wine    yeast strains leads to substantial changes in By-product formation    and to a stimulation of fermentation rate in stationary phase. Appl.    Environ. Microbiol. 65:143-149.

What is claimed is:
 1. A process for obtaining a variant yeast straincapable of producing, when compared to an ancestral yeast strain, moreglycerol and less ethanol during an alcoholic fermentation process, saidprocess comprising: a) culturing the ancestral yeast strain in a firstculture medium comprising a salt capable of causing an hyperosmoticstress to the ancestral yeast strain, wherein the ancestral yeast strainis cultured in increasing salt concentrations and under conditions toachieve glucose depletion in the first culture medium so as to obtain afirst cultured yeast strain; and b) culturing the first cultured yeaststrain in a second culture medium comprising the salt, wherein the firstcultured yeast strain is cultured at a fixed salt concentration andunder conditions to achieve glucose depletion in the second culturemedium so as to obtain the variant yeast strain; wherein the salt has acountercartion which is different than a sodium cation; and theconcentration of the salt in the second culture medium is higher thanthe concentration of the salt in the first culture medium.
 2. Theprocess of claim 1, wherein the concentration of the salt in the firstculture medium is between about 1.25 M and less than about 2.4 M and/orin the second culture medium is at least about 2.4 M.
 3. The process ofclaim 1 or 2, further comprising, at step a), increasing the saltconcentration weekly or monthly.
 4. The process of any one of claims 1to 3, wherein the first culture medium comprises glucose and the processfurther comprises, at step a), culturing the ancestral yeast strain inthe first culture medium in decreasing glucose concentrations, whereinthe concentration of glucose is preferably decreased weekly or monthlyand wherein the concentration of glucose in the first culture medium ismore preferably between about 14.0% and about 8.0% (w/v) with respect tothe total volume of the first culture medium.
 5. The process of any oneof claims 1 to 4, wherein the second culture medium comprises glucoseand the process further comprises, at step b), culturing the firstcultured yeast at a fixed glucose concentration and wherein the fixedglucose concentration of the second culture medium is preferably 8.0%(w/v) with respect to the total volume of the second culture medium. 6.The process of any one of claims 1 to 5, further comprising matinghaploid spores of the variant yeast strain to obtain a variant hybridstrain.
 7. The process of any one of claims 1 to 6, wherein the salt hasa potassium countercation.
 8. The process of claim 7, wherein the saltis KCl.
 9. The process of any one of claims 1 to 8, wherein the variantyeast strain is from a Saccharomyces species and preferably from a genusselected from the group consisting of Saccharomyces arboricolus,Saccharomyces eubayanus, Saccharomyces bayanus, Saccharomycescerevisiae, Saccharomyces kudriadzevii, Saccharomyces mikatae,Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomycescarsbergensis, Saccharomyces uvarum and inter-species hybrids.
 10. Avariant yeast strain capable of producing, when compared to an ancestralyeast strain, more glycerol and less ethanol in an alcoholicfermentation process, said variant yeast being obtained by the processof any one of claims 1 to
 9. 11. The variant yeast strain of claim 10for making a fermented product, preferably wine and more preferably ared wine.
 12. A variant yeast strain deposited at Institut Pasteur, onJan. 9, 2014, under accession number Collection Nationale des Culturesdes Microorganismes (CNCM) I-4832.
 13. A variant yeast strain depositedat Institut Pasteur, on Oct. 18, 2012 under accession number CollectionNationale des Cultures des Microorganismes (CNCM) I-4684.
 14. A variantyeast strain deposited at Institut Pasteur, on Oct. 18, 2012 underaccession number Collection Nationale des Cultures des Microorganismes(CNCM) I-4685.
 15. A variant yeast strain deposited at Institut Pasteur,on Jan. 28, 2015 under accession number Collection Nationale desCultures des Microorganismes (CNCM) I-4952.
 16. A process for making afermented product, said process comprising contacting the variant yeaststrain of any one of claims 10 to 15 with a fermentable source ofnutrients.
 17. The process of claim 16, wherein the fermented produce iswine, preferably red wine, and the fermentable source of nutrients is agrape must.